I. Origins of Allergic Disease Over 100 million people worldwide suffer from birch pollen allergy. However, identification of molecular determinants driving the allergic responses to Bet v 1, the major birch pollen allergen, remains elusive. In collaboration with a group in Salzburg, Austria, we examined the pollen microbiota and investigated the allergenicity of Bet v 1 upon interaction with pollen-derived compounds. At NIEHS we studied the binding of Bet v 1 to pollen derived compounds, which increased protein stability but did not induce allergic sensitization in vivo, even in the presence of mircrobial agonists. In contrast, birch pollen extracts (even those depleted of Bet v 1) strongly promoted an allergic type immune response. This finding indicates that the allergy polarizing potential of birch pollen extract is Bet v 1 independent. Sensitization to Bet v 1 is induced by an as-yet-undetermined pollen compound or mechanism in the pollen environment. These data suggest that sensitization is not exclusively linked to the intrinsic properties of individual proteins. II. Characterization of Allergens Dermatophagoides pteronyssinus (DP) and Dermatophagoides farinae (DF) are highly similar disease-associated mites with frequently overlapping geographic distributions. A draft genome of DP was assembled to identify candidate allergens in DP homologous to those in DF, investigate allergen isoforms, and facilitate comparisons with related Acari. The predicted size of the DP nuclear genome is 52.5 Mb. A predicted protein set of 19,368 proteins was identified, including all 19 currently recognized allergens from this species. Related proteins for 12 allergens established for DF were found. Some new isoforms were observed in both species suggesting that these isoforms pre-dated speciation. The high quality of both genomes allowed an examination of how allergen orthologs are physically clustered in the genome. In conclusion, candidate allergens in DP were identified to facilitate future serological studies. While DP and DF are highly similar genetically, species-specific allergen isoforms exist to facilitate molecular differentiation. Since the discovery that the major dust mite allergen Der p 1 is a cysteine protease, the role of proteolytic activity in allergic sensitization has been explored. There are many allergens with proteolytic activity; however, exposure from dust mites is not limited to allergens. To better understand the number of possible proteases in Dermatophagoides pteronyssinus (DP), genomic, transcriptomic and proteomic data from (DP) was mined. D. pteronyssinus has more proteases than the closely related Acari, Dermatophagoides farinae (DF) and Sarcoptes scabiei (SS). The group of proteases in D. pteronyssinus is found to be more highly transcribed than the norm for this species. Der p 1 surprisingly accounts for 22% of the total protease transcripts. In an analysis of protease stability, the group of protease allergens (Der p 1, Der p 3, Der p 6, and Der p 9) is found to be more stable than the mean. It is also statistically demonstrated that the protease allergens are simultaneously more highly expressed and more stable than the group of D. pteronyssinus proteases being examined, consistent with common assumptions about allergens in general. There are several significant non-allergen outliers from the normal group of proteases with high expression and high stability that should be examined for IgE binding. This study compiled the first holistic picture of the D. pteronyssinus degradome to which humans may be exposed. III. Adaptive Immune response The defining characteristic of allergy is the generation of IgE antibodies, which leads to patient symptoms. We wish to probe more fundamental properties of the antibody response. It is suggested that by better understanding and characterizing the antibodies of all types new treatment modalities, or safer therapies can be developed. Previously, we determined the first structure of the major peanut allergen Ara h 2. Ara h 2 is recognized by more than 90% of peanut allergic patients and sensitivity to Ara h 2 is measurable risk factor for peanut induced anaphylaxis. We have initiated a collaboration with Wayne Shreffler at Harvard University who has been studying the antibody production of peanut allergic patients in response to oral immunotherapy. By isolating B-cells from the patients and sequencing immunoglobulin genes, they found evidence that separate patients were honing in on similar regions of Ara h 2. We have received 5 of these antibody clones and are working to determine structures of antibody fragments in complex with Ara h 2. So far, we tested the production of all 5, and have scaled up production. The epitope information derived from these complexes will be useful in understanding the targets of the adaptive immune response of patients during peanut immunotherapy. Directly examining human IgE in complex an allergen is technically challenging for a number of reasons. First of all, the memory cells that make IgE are extremely rare in sera. Scott Smith at Vanderbilt University has recently developed a technique to clone these rare cells and produce full length human IgE. We have acquired 4 human monoclonal IgE antibodies against the major dust mite allergen Der p 2 to study as a collaborative project. In the past year we have been measuring the interactions of the human IgE with Der p 2 via NMR technologies. We hope that this pioneering technique could be generalized to study the response to other allergens, and may be applicable to other antibody types besides IgE. The results will help us better understand the human immune response to allergens like Der p 2 with a hope using the information to design hypo-allergens that will improve allergy immunotherapy, better known as allergy-shots.